Restoring colistin sensitivity in colistin-resistant Salmonella and Escherichia coli: combinatorial use of berberine and EDTA with colistin.

The appearance and prevalence of multidrug-resistance (MDR) Gram-negative bacteria (GNB) have limited our antibiotic capacity to control bacterial infections. The clinical efficacy of colistin (COL), considered as the "last resort" for treating GNB infections, has been severely hindered by its increased use as well as the emergence and prevalence of mobile colistin resistance (MCR)-mediated acquired drug resistance. Identifying promising compounds to restore antibiotic activity is becoming an effective strategy to alleviate the crisis of increasing MDR. We first demonstrated that the combination of berberine (BBR) and EDTA substantially restored COL sensitivity against COL-resistant Salmonella and Escherichia coli. Molecular docking indicated that BBR can interact with MCR-1 and the efflux pump system AcrAB-TolC, and BBR combined with EDTA downregulated the expression level of mcr-1 and tolC. Mechanically, BBR combined with EDTA could increase bacterial membrane damage, inhibit the function of multidrug efflux pump, and promote oxidative damage, thereby boosting the action of COL. In addition, transcriptome analysis found that the combination of BBR and EDTA can accelerate the tricarboxylic acid cycle, inhibit cationic antimicrobial peptide (CAMP) resistance, and attenuate Salmonella virulence. Notably, the combination of BBR and EDTA with COL significantly reduced the bacterial load in the liver and spleen of a mice model infected with Salmonella. Our findings revealed that BBR and EDTA can be used as adjuvants collectively with COL to synergistically reverse the COL resistance of bacteria. IMPORTANCE: Colistin is last-resort antibiotic used to treat serious clinical infections caused by MDR bacterial pathogens. The recent emergence of transferable plasmid-mediated COL resistance gene mcr-1 has raised the specter of a rapid worldwide spread of COL resistance. Coupled with the fact of barren antibiotic development pipeline nowadays, a critical approach is to revitalize existing antibiotics using antibiotic adjuvants. Our research showed that berberine combined with EDTA effectively reversed COL resistance both in vivo and in vitro through multiple modes of action. The discovery of berberine in combination with EDTA as a new and safe COL adjuvant provides a therapeutic regimen for combating Gram-negative bacteria infections. Our findings provide a potential therapeutic option using existing antibiotics in combination with antibiotic adjuvants and address the prevalent infections caused by MDR Gram-negative pathogens worldwide.

A Clinical Study of Platelet-Rich Fibrin Combined With Autologous High-Density Fat Transplantation in Augmentation Rhinoplasty.

OBJECTIVE: This study was designed to analyze the clinical effect of autologous fat-granule transplantation in augmentation rhinoplasty and explore methods to improve the fat retention rate. METHODS: A total of 70 enrolled patients were randomly divided into 2 groups: the platelet-rich fibrin (PRF) combined with high-density fat transplantation group (combined group) and the conventional fat-granule transplantation group (control group; n = 35 in each group). In the combined group, an appropriate amount of autologous fat was extracted and centrifuged, and the lower layer of high-density fat was taken and mixed with PRF isolated from whole blood for autotransplantation. In the control group, only fat was extracted and centrifuged for transplantation. The patients were followed up with for more than one year to observe the short- and long-term effects, complications, safety, and patient satisfaction. RESULTS: Six months after the operation, the nasal shape was stable, the contour was higher and more stereoscopic than before, the average increase of nasal height was 3.0 mm in the combined group and 2.0 mm in the control group. No complications, such as fat embolism, infection, or necrosis occurred during the 1-year follow-up. The satisfaction rate between the 2 groups has statistical significance (P < .05). CONCLUSION: Overall, PRF combined with autologous high-density fat transplantation is simple to perform, has a significantly increased fat-retention rate than the control group, and has stable long-term effects without obvious adverse reactions. A sufficient amount of fat and PRF transplantation can achieve a good orthopedic effect. Thus, this method can be widely used in clinical augmentation rhinoplasty.

Clinical Application of a Bird-Beak-Type Z-Shaped Asymmetrical Flap in the Reconstruction of the Inner Canthus.

Objective: To introduce a new surgical method for the repair of a large inner canthus combined with tissue loss at the inner canthal angle of the eye by using a bird-beak-type z-shaped asymmetrical flap and to summarize its clinical effect. Method: A total of 56 patients with a large inner canthus were randomly selected, and a bird-beak-type z-shaped asymmetrical flap was used on the nasal side of the lower eyelid to repair and reconstruct the inner canthal folds. The inner canthal point was located according to physiological aesthetics. The short and long arms of the z-shaped asymmetrical flap were separated, replaced, fixed, and shaped to reconstruct the skin folds of the inner canthus and restore its aesthetic morphology. Results: All incisions after surgery achieved primary healing, and all 56 cases were followed up for 6-20 months (average 8.6 months). The caruncula lacrimalis was moderately exposed, the inner canthal angles possessed a natural appearance, and the results of the surgery were satisfactory. Five patients developed scar hyperplasia within one month after surgery, and arnica gel was applied topically for 3-6 months until the scar faded or disappeared, but no obvious scars were seen in the surgical area of the remaining patients. In two patients, the internal canthi were asymmetrical, but this improved after adjustment. Conclusion: Repair of a large inner canthus and tissue loss at the inner canthal angle of the eye using a bird-beak-type z-shaped asymmetrical flap is a simple operation, resulting in minimal trauma. Postoperatively, the inner canthal angle possessed a natural appearance with no obvious scarring.

Molecular Characterization of a Novel Integrative Conjugative Element ICEHpa1 in Haemophilus parasuis.

ICEHpa1 was identified in the genome of a serovar 8 Haemophilus parasuis ST288 isolate YHP170504 from a case of swine lower respiratory tract infection. The aim of the present study was to characterize the integrative conjugative element ICEHpa1 and its multiresistance region. Susceptibility testing was determined by broth microdilution and the complete ICEHpa1 was identified by WGS analysis. The full sequence of ICEHpa1 was analyzed with bioinformatic tools. The presence of ICEHpa1, its circular intermediate and integration site were confirmed by PCR and sequence analysis. Transfer of ICEHpa1 was confirmed by conjugation. ICEHpa1 has a size of 68,922 bp with 37.42% GC content and harbors 81 genes responsible for replication and stabilization, transfer, integration, and accessory functions, as well as seven different resistance genes [bla Rob- 3, tet(B), aphA1, strA, strB, aac(6)'-Ie-aph(2')-Ia, and sul2]. Conjugation experiments showed that ICEHpa1 could be transferred to H. parasuis V43 with frequencies of 6.1 × 10-6. This is the first time a multidrug-resistance ICE has been reported in H. parasuis. Seven different resistance genes were located on a novel integrative conjugative element ICEHpa1, which suggests that the ICEHpa1 is capable of acquiring foreign genes and serving as a carrier for various resistance genes.

Application effect of lattice laser in facial rejuvenation: A protocol for systematic review and meta-analysis.

BACKGROUND: Various techniques have been applied in facial rejuvenation and lattice laser is the most accepted. However, the application effect of lattice laser in facial rejuvenation is unclear. This study aims to evaluate the application effect of lattice laser in facial rejuvenation. METHODS: Randomized controlled trials of lattice laser in facial rejuvenation will be searched in PubMed, EMbase, Web of Science, Cochrane Library, China National Knowledge Infrastructure, WanFang, the Chongqing VIP Chinese Science and Technology Periodical Database, and China biomedical literature database from inception to July 2020. And Baidu Scholar, International Clinical Trials Registry Platform, Google Scholar, and Chinese Clinical Trials Registry will be searched to obtain more relevant studies comprehensively. Two researchers will perform data extraction and risk of bias assessment independently. Statistical analysis will be conducted in RevMan 5.3. RESULTS: This study will sum up the present evidence so far by exploring the application effect of lattice laser in facial rejuvenation. CONCLUSIONS: The findings of the study will provide helpful evidence for the application effect of lattice laser in facial rejuvenation, promoting clinical practice, and further scientific research. ETHICS AND DISSEMINATION: The private information from individuals will not publish. This systematic review also will not involve endangering participant rights. Ethical approval is not required. The results may be published in a peer-reviewed journal or disseminated in relevant conferences. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/QF6H5.

Characterization of pTS14, an IncF2:A1:B1 Plasmid Carrying tet(M) in a Salmonella enterica Isolate.

The objective of this study was to explore the genetic and biological features of the tet(M)-harboring plasmid pTS14 in Salmonella enterica strain S14 isolated from a chicken fecal sample. Plasmid pTS14 was identified by conjugation, S1-pulsed-field gel electrophoresis (PFGE), Southern hybridization, and plasmid sequencing. The biological characteristics of pTS14 were assessed via stability, growth kinetics, and starvation survival experiments. Strain S14, belonging to ST3007, harbored a 119-kb tet(M)-bearing IncF2:A1:B1 conjugative plasmid pTS14. The plasmid pTS14 contained a novel transposon Tn6709 with the genetic structure IS26-tnpA1-tnpA2-Δorf13-LP-tet(M)-tnpX-ΔtnpR-IS26, and the resistance genes tet(B), tet(D), strAB, sul2, and bla TEM-1b. In addition, pTS14 was found to be highly stable in the recipient strain E. coli J53. The transconjugant TS14 exhibited a higher survival ratio than E. coli J53 under permanent starvation-induced stress. The tet(M)-bearing IncF2 epidemic plasmid lineage may accelerate the dissemination of tet(M) and other genes by coselection, which could constitute a potentially serious threat to clinical treatment regimens.

Improved stability of KGF by conjugation with gold nanoparticles for diabetic wound therapy.

Aim: Diabetic wound healing is seriously interrupted, and administration of KGF for wound treatment is restricted by its inherent instability. We aim to develop an ideal way toward KGF stabilization, thus improving diabetic wound healing. Materials & methods: We conjugated KGF with gold nanoparticles (GNPs) and determined the stability and binding affinity. Biological effects of conjugates (KGF-GNPs) were evaluated in vitro and in an animal model. Results: KGF-GNPs revealed high stability under hostile circumstances because of the preserved secondary structure and possessed elevated binding affinity to KGF receptor. Moreover, application of KGF-GNPs contributed to accelerated wound recovery in diabetic rats, including re-epithelialization and contraction. Conclusion: KGF-GNPs were promising for future clinical application for diabetic wound therapy.

Effects of Myeloperoxidase on Methicillin-Resistant Staphylococcus aureus-Colonized Burn Wounds in Rats.

Objective: To achieve better therapeutic results in burn wound infections and to examine alternatives to antibiotics, we designed this study to elaborate the role of myeloperoxidase (MPO) on infected burn wounds in rats. Approach: We compared chemical properties as well as bacteriostatic ability of MPO in different concentrations with NeutroPhase. Subsequently, we applied MPO (MPO group), NeutroPhase (NeutroPhase group), NaCl+H2O2 (NaCl+H2O2 group), or NaCl (control group) on rat dorsal burn wounds inoculated with methicillin-resistant Staphylococcus aureus (MRSA). Their effects on MRSA-colonized wounds were evaluated by microscopy, histologic section, and Western blot. Results: MPO produced more H+ and HClO-, leading to a more acidic environment. Moreover, MPO inhibited the growth of MRSA more intensely after 6 h of inoculation ex vivo. In vivo the open wound rate in the MPO group was significantly lower, while the contraction rate and epithelialization rate of MPO group were higher than that of the control group, NaCl+H2O2 group, and NeutroPhase group on day 20. The hematoxylin and eosin staining of MPO group showed better wound healing than other groups. More vascular endothelial growth factor (VEGF) was expressed in wound tissue of MPO group by Western blot. Innovation: This is the first study to use MPO for MRSA-colonized burn wound therapy. Conclusion: MPO displayed more effective bacteriostatic ability, possibly beneficial for MRSA-colonized wound healing.

KGF-1 accelerates wound contraction through the TGF-ß1/Smad signaling pathway in a double-paracrine manner.

KGF-1 plays an important role in the wound healing process. Loss of the KGF-1 gene in diabetic mice attenuated the process of wound contraction, suggesting that KGF-1 contributes to wound contraction. However, the mechanism remains unclear. To investigate the role of KGF-1 in diabetic wound contraction, we established a keratinocyte-fibroblast co-culture system. Concentrations of transforming growth factor ß1 (TGF-ß1) in conditioned supernatant treated with KGF-1 (KGF-1 group), tk;4KGF-1-neutralizing antibody (anti-KGF-1 group), TGF-ß1 (TGF-ß1tk;1 group), KGF-1 and TGF-ß1-neutralizing antibody (KGF-1 + anti-TGF-ß1 group) were tested by ELISA. Conditioned medium was added to fibroblast-populated collagen lattice (FPCL) to investigate the effect of KGF-1 on fibroblastqj contraction. TGF-ß1, Col-I, p-Smad2, p-Smad3, and α-smooth muscle actin (α-SMA) were examined by Western blotting. A diabetic rat wound model was utilized to evaluate wound morphology, histology, immunohistochemistry, and protein expression in wound tissue after treatment with KGF-1. ELISA assays revealed that the concentration of TGF-ß1 in the conditioned supernatant in the KGF-1 group was significantly higher. The contractile capacity of FPCL stimulated by conditioned medium derived from the KGF-1 group was significantly elevated; however, the contractile activity of FPCL induced by KGF-1 was attenuated by TGF-ß1-neutralizing antibody. The Western blot results suggest that KGF-1 is able to stimulate TGF-ß1 activation with increased Col-I, p-Smad2, p-Smad3, and α-SMA expression. Diabetic wounds treated with KGF-1 had a higher degree of contraction with significantly higher expression of TGF-ß1, Col-I, p-Smad2, p-Smad3, and α-SMA. Our findings demonstrate that KGF-1 promotes fibroblast contraction and accelerates wound contraction via the TGF-ß1/Smad signaling pathway in a double-paracrine manner.

A G-quadruplex based fluorescent oligonucleotide turn-on probe towards iodides detection in real samples.

A basket-type G-quadruplex (GQ) fluorescent oligonucleotide (OND) probe is designed to detect iodides dependent on thymine-Hg(II)-thymine (T-Hg(II)-T) base pairs and the intrinsic fluorescence quenching capacity of GQ. In the presence of Hg(II) ions (Hg2+), the two hexachloro-fluorescein-labeled ONDs form a hairpin structure and the fluorophores are dragged close to the GQ, leading to fluorescence quenching of the probe due to photoinduced electron transfer. Upon addition of iodide anions, Hg2+ are extracted from T-Hg(II)-T complexes which attributes to the stronger binding with iodide anions, resulting in the fluorescence recovery. Through performing the fluorescence quenching and recovery processes, this probe developed a fluorescence turn-on sensor for iodide anions determination over a linear range of 20-200nmol/L with a limit of detection of 5nmol/L. The practical use of the turn-on technology was demonstrated by its application in determination of iodides in water, food, pharmaceutical products and biological samples.

Molecular recognition and binding of beta-lactamase II from Bacillus cereus with penicillin V and sulbactam by spectroscopic analysis in combination with docking simulation.

The molecular recognition and binding interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) at 277 K were studied by spectroscopic analysis and molecular docking. The results showed that a non-fluorescence static complex was separately formed between Bc II and two ligands, the molecular ratio of Bc II to PV or Sul was both 1:1 in the binding and the binding constants were 2.00 × 106 and 3.98 × 105 (L/mol), respectively. The negative free energy changes and apparent activation energies indicated that both the binding processes were spontaneous. Molecular docking showed that in the binding process, the whole Sul molecule entered into the binding pocket of Bc II while only part of the whole PV molecule entered into the pocket due to a long side chain, and electrostatic interactions were the major contribution to the binding processes. In addition, a weak conformational change of Bc II was also observed in the molecular recognition and binding process of Bc II with PV or Sul. This study may provide some valuable information for exploring the recognition and binding of proteins with ligands in the binding process and for the design of novel super-antibiotics.

Spectroscopy analysis and molecular dynamics studies on the binding of penicillin V and sulbactam to beta-lactamase II from Bacillus cereus.

The molecular recognition and interaction of beta-lactamase II from Bacillus cereus (Bc II) with penicillin V (PV) and sulbactam (Sul) especially conformational changes of Bc II in the binding process were studied through spectroscopy analysis in combination with molecular dynamics (MD) simulation. The results show that in the binding process, a new coordination bond is observed between the Zn2 of Bc II and the carboxyl-O of PV or Sul by replacing His204. Electrostatic interaction between Zn2 and the ligand provide main driving force for the binding affinity. Compared with apo Bc II, there are mainly four loops showing significant conformational changes in ligand-bound Bc II. A weak conformational transformation from ß-sheets to random coils is observed in the loop2 of ligand-bound Bc II. The conformational transformation may depend on the functional group and binding pose of the ligand, giving the binding pocket greater flexibility and accordingly allowing for an induced fit of the enzyme-ligand binding site around the newly introduced ligand. The change in the loop2 of ligand-bound Bc II may lead to the opening of the binding pocket of Bc II. Therefore, loop2 can be considered a gate for control of ligand access in Bc II, hence its dynamic response should be considered in new drug design and development.

Inhibitory action of pristimerin on hypoxia­mediated metastasis involves stem cell characteristics and EMT in PC-3 prostate cancer cells.

The aim of the present study was to investigate whether pristimerin affects the bone metastasis, stem cell characteristics and epithelial-mesenchymal transition (EMT) of prostate cancer (PCa) PC-3 cells subjected to hypoxia. The PC-3 cells were cultured under hypoxia or normoxia for 48 h and were then treated with increasing concentrations of pristimerin from 0 to 0.8 µmol/l, under normoxia. Hypoxia­inducible factor-1α (HIF-1α) was detected by western blotting. Proliferation was assessed with the CCK-8 assay. Transwell invasion assay was used to analyze the potency of invasion. Stem cell characteristics were detected by sphere formation, colony formation assay and western blotting, including CD44, KLF4, OCT4 and AGO2, which are stem cell characteristic-related markers. EMT was confirmed by the expression changes of EMT-related markers, including N-cadherin, fibronectin, vimentin and ZEB1, which were evaluated by western blotting. The addition of pristimerin to the medium reduced the hypoxia-induced PC-3 cell proliferation in a dose-dependent manner. Pristimerin effectively inhibited hypoxia­induced invasion of the PCa cells in vitro. Moreover, the treatment of cells with pristimerin induced the reversal of hypoxia-induced stem cell characteristics and EMT, which was confirmed by sphere formation, colony formation assay and the expression changes of CSC- and EMT-related markers. The reversal of hypoxia­induced stem cell characteristics and EMT in the PCa cells by low-dose pristimerin was dose­dependent. These results showed that treatment with pristimerin may be a potential strategy for the suppression of hypoxia-induced metastasis through the reversal of hypoxia-induced stem cell characteristics and EMT in cancer cells, which justifies the potential use of pristimerin as a practical chemopreventive approach for patients with PCa.